Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the ability of all eukaryotic cells to function, grow and communicate. Fully one-third of the eukaryotic proteome is targeted to the membrane compartments that comprise the secretory pathway. These proteins must be faithfully delivered to these organelles after synthesis and folding in the endoplasmic reticulum (ER). Proteins that fail to fold correctly are prevented from leaving the ER and targeted for destruction, a process known as ER quality control. We study the relationship between protein folding within the ER and the ability of nascent proteins to be captured into ER-derived transport vesicles for downstream delivery. We aim to uncover the rules that govern whether a protein is a substrate for forward transport, a question that lies at the heart of ER quality control. We use the model organism, Saccharomyces cerevisiae, to study this problem using a combination of genetic and biochemical approaches, focusing on the biogenesis of the yeast ABC transporter, Yor1p, a drug pump that confers resistance to the toxin, oligomycin. Yor1p is broadly related to the human cystic fibrosis transmembrane conductance regulator, CFTR, which causes cystic fibrosis when mutated. Expression of disease-related misfolded variants of Yor1p have permitted the discovery of components involved in biogenesis of this family of membrane proteins through genome-wide phenotypic analysis of oligomycin resistance. This approach led us to a set of ER membrane proteins that function in regulating the fate of folded versus misfolded forms of Yor1p at distinct stages. The EMC complex seems to act very early during protein synthesis to stabilize an aberrant variant of Yor1p; the putative export factor, Erv14p, functions to promote efficient ER egress of both wild-type and mutant forms of Yor1p. The current research proposal consists of three specific aims. (1) To determine the mechanism by which the EMC complex and related components influence the biogenesis of nascent membrane proteins. (2) To further define the cellular machinery that acts in the ER during biogenesis of membrane proteins to regulate ER quality control and forward traffic. (3) To characterize the complex mechanisms that drive Erv14p- and Sec24p-dependent export from the ER. Using these approaches, we seek to gain detailed insight into the molecular mechanisms that underlie the ER quality control checkpoint. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease that are impacted by protein folding lesions that preclude deployment within the secretory pathway. PUBLIC HEALTH RELEVANCE: We study the process of protein quality control in the eukaryotic endoplasmic reticulum to understand how cells make decisions about the fate of proteins to prevent the deployment of aberrant, potentially toxic molecules. This work has important implications for the growing number of diseases that are caused by aberrant folding, trafficking and deployment of secretory proteins, including cystic fibrosis, familial heart disease and lung disease.